JP2011217701A - High-purity epilactose and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for producing highly purified epilactose by separating epilactose from lactose which is isomerized sugar.SOLUTION: The method for producing high-purity epilactose includes allowing β-galactosidase to act on a solution containing lactose and epilactose, chromatographically separating a monosaccharide fraction and a disaccharide fraction from the solution on which the β-galactosidase has acted, and collecting epilactose from the disaccharide fraction.

Description

  The present invention relates to high-purity epilactose and a method for producing the same. Specifically, by selectively separating and recovering epilactose from a mixture of lactose and epilactose, highly purified epilactose can be efficiently recovered. The present invention relates to a production method and high-purity epilactose obtained by this method.

  Epilactose is a disaccharide in which galactose and mannose are linked by β-1,4 from the non-reducing end, and is a rare sugar produced in a trace amount by heat sterilization of milk or alkali isomerization of lactose (see Non-Patent Documents 1 and 2). ).

  In recent years, physiological function analysis on epilactose has been promoted, and it has been clarified that the sugar has functions such as bifidobacteria growth activity (see Non-patent Document 3) and calcium absorption promoting activity (see Non-Patent Document 4). .

  The epilactose can be synthesized enzymatically from lactose under mild conditions by utilizing the 2-epimerase activity of cellobiose 2-epimerase (see Non-Patent Document 5). Usually, the amount of epilactose of the reaction product by the enzyme reaction is as low as about 30% by mass in the total sugar solid content, and a large amount of unreacted lactose remains in the reaction product. Therefore, in order to obtain highly purified epilactose, it is necessary to separate lactose and epilactose from the reaction product and selectively recover epilactose.

  For example, Patent Document 1 employs preparative liquid chromatography using Shodex SP0810 manufactured by Shodex, which is a ligand exchange column, as a method for the above-described purification.

  On the other hand, for increasing the purity of rare sugars, for example, Patent Document 2 proposes to employ a simulated moving bed chromatographic separation apparatus. The simulated moving bed type chromatographic separation apparatus mentioned here typically includes a large number of packed towers filled with an adsorbent having a selective adsorption ability with respect to a specific component of two or more components contained in the stock solution. By connecting in series with piping, and connecting the last filling tower and the foremost filling tower with piping, a device is formed as a system of packed towers that are connected endlessly, supplying raw solution, eluent Supply, extraction of non-adsorbed liquid containing a high amount of substances with low adsorption capacity to the adsorbent, and extraction of adsorbed liquid containing a large amount of substances with high adsorption capacity in the adsorbent while maintaining a constant positional relationship. Is moved to the downstream side in the circulation direction in the system over time, and thus the apparatus performs the same function as the adsorbent moves without actually moving the adsorbent.

  For example, if the sugar is highly crystalline, such as glucose or sucrose, the solution containing these sugars is concentrated to a concentration exceeding the solubility, or the crystal is precipitated by cooling or adding seed crystals. It is possible to dramatically increase the purity of carbohydrates. At this time, if necessary, the crystal is dissolved again and further crystallized (recrystallized), so that further high purity can be achieved.

  Moreover, highly purified saccharides can also be obtained by combining liquid chromatography and crystallization. For example, in Patent Document 3, in order to increase the purity of nystose, which is a kind of fructooligosaccharide, a fructooligosaccharide mixed solution (mixed solution of monosaccharides to 6 sugars) is rich in nisose, which is a tetrasaccharide, by liquid chromatography. A method of crystallizing nystose by obtaining a fraction and cooling it is disclosed.

WO2008 / 062555 JP 2001-354690 A JP-A-6-339388

Milchwissenshaft 1980; 35 (1): 5-8 Milchwissenshaft 1981; 36 (9): 533-536 J. et al. Dairy Sci. 2008; 91: 4518-4526 J. et al. Agric. Food Chem. 2008; 56: 10340-10345 Appl. Microbiol. Biotechnol. 2008; 79: 433-441

  However, in order to separate epilactose from lactose, which is its isomerized sugar, to achieve high purity, the above conventional methods have the following problems.

  In the ligand exchange column used in the method described in Patent Document 1, lead that is not approved for use in food is used for the ligand. Therefore, since epilactose purified using this column may contain lead, its application to food and drink is limited. However, according to the study by the inventors of the present application, other ligands that can be used for food and drink cannot separate epilactose from the mixture with lactose.

  In order to achieve high purity by the simulated moving bed chromatographic separation apparatus described in Patent Document 2, it is necessary to use an adsorbent having a selective adsorption ability with respect to the substance to be separated, either epilactose or lactose. No adsorbent having a selective adsorption capacity on only one side has been found other than the above-described ligand exchange column using lead.

  As described above, crystallization is also known as a means for achieving high purity. According to the study by the present inventors, lactose has lower solubility than epilactose, so that lactose can be preferentially precipitated by crystallization. However, studies by the present inventors have revealed that there is a limit to the purity that can be achieved only by subjecting a mixed solution of lactose and epilactose to crystallization treatment. This is because epilactose and lactose are isomers and the structure is close, and as the concentration of epilactose increases as crystallization proceeds, the lactose inhibits crystallization of lactose. The inventors speculate. Therefore, there is a need to find a high-purity means that can replace the crystallization process, or a high-purity means that can be implemented together with the crystallization process.

  In addition, there have been many reports on methods for purifying saccharides, and methods using activated carbon column chromatography or gel filtration chromatography are also known. However, activated carbon column chromatography is inferior in workability because it is difficult to regenerate activated carbon, which is a carrier, and it is necessary to replace the carrier every fraction. Both activated carbon column chromatography and gel filtration chromatography are practically limited to fractionation at the laboratory level of sample preparation on the order of several grams because the amount of sample that can be loaded at one time is small.

  Accordingly, an object of the present invention is to provide a means for producing highly purified epilactose by separating epilactose from its isomerized sugar, lactose, and more specifically, cellobiose 2-epimerase. An object of the present invention is to provide a means for producing highly purified epilactose applicable to food and drink use from a mixed solution of epilactose and lactose obtained by allowing sucrose to act on lactose.

As a result of intensive studies to achieve the above object, the inventors of the present application have shown that β-galactosidase, which is a lactose hydrolase, exhibits hydrolysis activity against epilactose, which is an isomerized sugar of lactose. Since there is no (or almost no indication), if β-galactosidase is allowed to act on a mixture of lactose and epilactose and then the monosaccharide fraction and the disaccharide fraction are chromatographed, the hydrolyzate of lactose ( It was newly found that epilactose can be obtained as a disaccharide fraction by separating galactose and glucose). Many adsorbents that do not use lead are known as long as they are capable of separating the above monosaccharides and disaccharides. Therefore, highly purified epilactose that can be applied to food and drink by using these adsorbents. Can be obtained.
The present invention has been completed based on the above findings.

That is, the above object has been achieved by the following means.
[1] allowing β-galactosidase to act on a solution containing lactose and epilactose;
Chromatographic separation of a monosaccharide fraction and a disaccharide fraction from the solution in which the β-galactosidase is allowed to act; and
Recovering epilactose from the disaccharide fraction,
The manufacturing method of the high purity epilactose characterized by including this.
[2] The production method according to [1], further comprising subjecting a solution containing lactose and epilactose to a crystallization treatment and separating the precipitated crystallized product before allowing β-galactosidase to act.
[3] The production method according to [1] or [2], wherein the chromatographic separation is performed using an alkali metal type or alkaline earth metal type cation exchange resin.
[4] The production method according to [3], wherein the ion exchange resin is a sodium-type cation exchange resin.
[5] The production method according to any one of [1] to [4], wherein the chromatographic separation is performed by simulated moving bed column chromatography.
[6] The production method according to any one of [1] to [5], wherein the solution containing the lactose and epilactose is obtained by an enzymatic reaction of cellobiose 2-epimerase using lactose as a substrate.
[7] High purity epilactose obtained by the method according to any one of [1] to [6].

  ADVANTAGE OF THE INVENTION According to this invention, the high purity epilactose applicable to food-drinks can be provided.

The chromatogram in Example 1 is shown. The chromatogram in Example 2 is shown. The change of the epilactose content by the concentration in Reference Example 1 is shown.

The present invention relates to a method for producing high-purity epilactose including the following steps.
Allowing β-galactosidase to act on a solution containing lactose and epilactose (hereinafter referred to as “hydrolysis step”);
Chromatographic separation of a monosaccharide fraction and a disaccharide fraction from the solution treated with β-galactosidase (hereinafter referred to as “chromatography separation step”); and
Recovering epilactose from the disaccharide fraction (hereinafter also referred to as “epilactose recovery step”).
Hereinafter, each of the above steps will be sequentially described.

Hydrolysis step epilactose (4-0-β-D-galactopyranosyl-D-mannose) is a lactose isomerism obtained by 2-epimerization of glucose at the reducing end of lactose with cellobiose 2-epimerase. Is the body. Therefore, the solution in which β-galactosidase is allowed to act in this step can be obtained by an enzymatic reaction of cellobiose 2-epimerase using lactose as a substrate. As cellobiose 2-epimerase, for example, one derived from Ruminococcus albus can be used, and an enzyme sample can be prepared from the culture solution of this strain according to a conventional method. In addition, it is advantageous to use a recombinant enzyme produced by recombinant Escherichia coli or Bacillus subtilis transformed with an expression vector into which a DNA encoding the enzyme is introduced, for easy preparation of the enzyme in large quantities. Regarding this point, Patent Document 1 can be referred to.

Although the reaction conditions of lactose and cellobiose 2-epimerase are not particularly limited, from the viewpoint of reaction efficiency, cellobiose 2-epimerase having an enzyme activity of 0.1 to 10 U with respect to 1 g of lactose solid content is added. It is preferable to do. Here, the activity of cellobiose 2-epimerase is defined as the amount of enzyme that produces 1 μmol of glucosyl mannose per minute under the following conditions and 1 U.
Method for measuring cellobiose 2-epimerase activity After adding 150 μl of an enzyme sample appropriately diluted with a mixture of 250 μl of 200 mM glycylglycine (pH 7.8) and 100 μl of 25 mg / ml cellobiose, the mixture was kept at 30 ° C., and then deactivated by boiling. The amount of released glucosyl mannose is quantified. Glucosyl mannose is quantified using HPLC. Specifically, quantification by HPLC can be performed under the following conditions. The cellobiose 2-epimerase activity shown in Examples described later is a value measured under the following conditions.
500 μl is collected from the reaction solution diluted 5-fold, applied to AG501-X8 column (φ4 × 10 mm) manufactured by Bio-Rad, and 90 μl is collected from the eluate and mixed with 10 μl of 2 mg / ml maltitol as an analysis sample. . Maltitol is used as an internal standard. As HPLC analysis conditions, Sugar SP0810 (φ8 × 300 mm) manufactured by Shodex was used as a column, the mobile phase was pure water, and the flow rate was set to 0.8 ml per minute. The sugar is detected by evaporation light scattering detection using ELD2000ES manufactured by Alltech Associates.

  The substrate concentration of the reaction solution is suitably about 1 to 20% (w / v), but about 10 to 20% (w / v) is preferable from the viewpoint of the treatment efficiency of the reaction solution. Since lactose dissolves as the isomerization reaction proceeds, lactose may not be completely dissolved at the start of the reaction.

  The pH and temperature during the reaction can be appropriately determined according to the characteristics of the enzyme used. In addition, the reaction time and the enzyme addition amount are closely related, and the reaction time can be adjusted by adjusting the enzyme addition amount as necessary. Specifically, the reaction can be performed for 10 to 90 hours at a temperature in the range of pH 3.0 to 10.0 and 15 ° C. to 85 ° C. Moreover, a well-known buffer solution can be used for pH adjustment.

  After a predetermined time, the reaction is terminated by inactivating the enzyme, for example, by heating the reaction solution. Usually, the amount of epilactose produced by the enzyme reaction is about 30% by mass in the total sugar solids, and the reaction solution contains the unreacted lactose together with the reaction product epilactose. Therefore, in the present invention, only the lactose is selectively hydrolyzed in a mixed solution of epilactose and lactose by utilizing the selective hydrolysis property of β-galactosidase. Thereby, epilactose can be isolated with high purity as a disaccharide fraction by subsequent chromatographic separation.

  The solution after the completion of the enzyme reaction can be directly subjected to a hydrolysis reaction with β-galactosidase. It is preferable for further increasing the purity of lactose. This is because the solubility of lactose is lower than that of epilactose, so that lactose can be preferentially precipitated (crystallized) and removed by crystallization treatment.

  When carrying out the above crystallization treatment, activated carbon may be added to the reaction solution of cellobiose 2-epimerase and lactose after completion of the reaction, and the mixture may be heated to about 50 to 100 ° C. and filtered. The solution can also be desalted with an ion exchange resin. Since components other than neutral sugars can be removed by these operations, lactose crystallization can be promoted. Moreover, said heating and filtration can also be implemented as a decoloring and deodorizing process.

  The crystallization treatment can be performed by a known method such as concentration, cooling, or seed crystal addition. The cooling temperature may be, for example, 0 to 5 ° C., and the cooling period may be about 1 day to 1 week. Concentration is preferably performed by concentration under reduced pressure, and the total sugar concentration after concentration is preferably 60% (w / v) or more from the viewpoint of lactose removal efficiency by crystallization. Further, since the operability decreases as the solution viscosity increases, the total sugar solid concentration after concentration is preferably 80% (w / v) or less from the viewpoint of operability. By removing the precipitated lactose crystals after the crystallization treatment, for example, a honey solution containing 60 to 75% by mass of epilactose per solid content can be obtained. In addition, the lactose crystals removed here can be dissolved again and used for epilactose production reaction.

  Then, β-galactosidase is allowed to act on a mixed solution of lactose and epilactose (the above reaction solution or honey solution obtained after crystallization treatment) to selectively select lactose in the mixed solution as a monosaccharide (galactose and glucose). ) Can be hydrolyzed. On the other hand, since the coexisting epilactose does not receive (or hardly receives) hydrolysis by β-galactosidase, a disaccharide fraction containing epilactose with high purity can be obtained by subsequent chromatographic separation.

  Β-galactosidase used for the hydrolysis is readily available as a commercial product. The origin is not particularly limited, but those formulated are advantageous for industrial use. Examples of commercially available product names include Lactores L3 manufactured by Daiwa Kasei Co., Ltd., which is β-galactosidase derived from Bacillus circulans.

  In the treatment with β-galactosidase, if the total sugar concentration in the solution is too high, the transglycosylation reaction due to β-galactosidase becomes prominent and the efficiency of fractionation decreases, and if it is too low, the efficiency of subsequent concentration decreases. To do. Considering the fractionation efficiency and the concentration efficiency, the treatment with β-galactosidase is preferably carried out by diluting the mixed solution with water to a solid content concentration of about 1 to 20% (w / v).

It is preferable that the addition amount of (beta) -galactosidase shall be about 1-50U per 1g of solid content from the point of the reaction efficiency of hydrolysis. In addition, let the enzyme activity of the said (beta) -galactosidase be the value measured with the following method.
Measurement of β-galactosidase activity To 1 mL of enzyme solution diluted to an appropriate concentration, 1 ml of 40 mM o-nitrophenyl-β-D-galactopyranoside dissolved in 0.1 M sodium acetate buffer (pH 6.0) was added, Hold at 40 ° C. for 10 minutes. To this is added 5 mL of a 2% by weight aqueous sodium carbonate solution and kept on ice for 10 minutes. The absorbance at 420 nm of the obtained solution is measured. The amount of enzyme that liberates 1 μmol of o-nitrophenol per minute under the above conditions is defined as 1 U.

  The pH and temperature in the hydrolysis reaction can be appropriately determined according to the characteristics of the enzyme (β-galactosidase) used. Moreover, since reaction time and the amount of enzyme addition are closely related, these can also be adjusted and set suitably. Specifically, β-galactosidase can be allowed to act at a temperature in the range of pH 4-8, 25-85 ° C. for about 1-24 hours. When the enzyme activity of β-galactosidase to be used is extremely high, there is a concern that epilactose may be decomposed if the reaction is continued excessively after the decomposition of lactose. Therefore, in order to improve the yield of epilactose, It is preferable to determine reaction conditions so that decomposition does not occur.

  The β-galactosidase treatment solution thus obtained is used for the subsequent operation after inactivating the enzyme by pH adjustment or heat treatment. For example, β-galactosidase can be inactivated by maintaining the temperature of the reaction solution at 80 ° C. or higher for about 1 hour. This reaction stop condition can be appropriately set according to the characteristics of the enzyme used. The treatment liquid after enzyme deactivation can be subjected to known treatments such as filtration and concentration.

Chromatographic separation step, epilactose recovery step Both lactose and epilactose are disaccharides, but lactose can be selectively hydrolyzed by treatment with β-galactosidase. Therefore, in the β-galactosidase treatment liquid obtained by the above process, since lactose is hydrolyzed into monosaccharides (galactose and glucose), most of the disaccharides contained are epilactose. Therefore, high purity epilactose can be obtained as the disaccharide fraction by chromatographic separation of the monosaccharide fraction and the disaccharide fraction.

  Before being subjected to chromatographic separation, it is preferable that the β-galactosidase treatment solution is subjected to activated carbon treatment and desalting treatment to remove components other than neutral sugars. This is because components other than the neutral sugar cause the ions coordinated to the ion exchange resin used for the chromatographic separation to drop out, and the fractionation efficiency is lowered. Both the activated carbon treatment and the desalting treatment can be performed by known methods.

  Any system that can separate monosaccharides and disaccharides can be used for the chromatographic separation without any limitation. The chromatographic separation may be performed by any of a fixed bed method, a moving bed method, and a simulated moving bed method, but from the viewpoint of separation efficiency, it is preferable to use simulated moving bed column chromatography. Simulated moving bed column chromatography is a system in which a large number of packed columns filled with an adsorbent having a selective adsorption capacity for a specific component of two or more components contained in a stock solution are connected in series by piping. By connecting the last filling tower and the front filling tower with a pipe, a device is formed as a system of packed towers that are connected endlessly as a whole, to supply raw solution, supply eluent, and adsorbent On the other hand, the position of the non-adsorbing liquid containing a high amount of substances with low adsorption capacity and the extraction of the adsorbing liquid containing a large amount of substances with high adsorption capacity in the adsorbed liquid are maintained over time while maintaining the same positional relationship. This is column chromatography that performs the same function as moving the adsorbent without actually moving the adsorbent by moving it sequentially downstream in the inner circulation direction. An apparatus for performing such chromatographic separation is known, and in the present invention, a commercially available simulated moving bed type chromatographic separation apparatus can be used. Among them, it is preferable to use an improved simulated moving bed chroma separator manufactured by Nippon Nersui Co., Ltd. Japanese Patent Publication No. 7-46097 can be referred to for the configuration.

  As the column to be used, in order to use the disaccharide fraction (high purity epilactose) after chromatographic separation for food and drink use, it is preferable to use a column that does not contain lead. As a column preferable from the above viewpoint, an alkali metal type or alkaline earth metal type cation exchange resin, more specifically, a sodium type or calcium type ion exchange resin can be mentioned, and a sodium type is preferable. The cation exchange resin is preferably a strongly acidic cation exchange resin having a sulfonic acid group as an exchange group. Examples of commercially available product names include Diaion (registered trademark) UBK530, which is a sodium-type strongly acidic cation exchange resin manufactured by Mitsubishi Chemical. Fractionation conditions such as sample injection volume, column size, column temperature, flow rate, etc. in chromatographic separation are not particularly limited, and can be set as appropriate so that the disaccharide fraction and the monosaccharide fraction are well separated. it can.

  Thereafter, a disaccharide fraction containing epilactose at a high concentration is collected from the fraction separated by the chromatographic separation. The sugar composition of the fraction to be separated can be analyzed by an HPLC method or the like, and a fraction with an increased epilactose content can be collected as a disaccharide fraction. High-purity epilactose can be recovered by subjecting the collected disaccharide fraction to treatment such as filtration, centrifugation, decolorization, desalting, and concentration as necessary. As described above, the amount of epilactose in the reaction product of cellobiose 2-epimerase enzymatically using lactose as a substrate is usually about 30% by mass in the total sugar solids, whereas according to the present invention, 40% by mass. It is possible to obtain epilactose with a high purity exceeding 80%, and furthermore, epilactose can be obtained with a high purity exceeding 80% by mass by carrying out a crystallization treatment. Further, according to the present invention, separation and purification using a column containing no lead is possible, so that the resulting epilactose is a novel epilactose that is substantially free of lead and highly purified. The above-mentioned epilactose can be used as it is or mixed with known additives for functional foods such as mineral absorption promoters, functional foods for promoting mineral absorption, constipation improving agents, lipid metabolism improving agents, lipid metabolism improving agents. It can be used as a functional food, a functional food for diabetic patients, and a low-calorie low sweetener. In addition, as for the details of application of epilactose as the functional food, paragraphs [0112] to [0123] of WO2008 / 062555 and Examples 5 to 11 of the publication can be referred to.

  The present invention will be further described below with reference to examples. However, the present invention is not limited to the embodiment shown in the examples. “%” Described below indicates mass% unless otherwise specified.

  The sugar composition in the following examples was calculated from the peak area by analyzing the sample by high performance liquid chromatography (HPLC). In HPLC, Sugar SP0810 (φ8 × 300 mm; Shodex) was used as a column, and the column temperature was set to 80 ° C. Pure water was used as the mobile phase, and the flow rate was set to 0.8 ml / min. A differential refractometer (PR-201 manufactured by Atago) was used for detecting sugar. Samples of 5% (w / v) were subjected to 10 μl analysis.

[Example 1]
5 mL of 1M potassium phosphate buffer solution (pH 6.3) is added to 0.5 L of 20% (w / v) lactose monohydrate (manufactured by Kanto Chemical Co., Ltd.), and luminococcus albus-derived cellobiose 2-epimerase is added to lactose solids. It added so that it might become 2U per 1g. This was held at 25 ° C. for 60 hours. When the epilactose content in the reaction solution after the enzyme reaction was measured by the method for analyzing sugar composition, the epilactose content in the total sugar solid content was 24.5%. That is, the epilactose purity at this stage was 24.5%.

Thereafter, the reaction solution after the enzyme reaction was diluted twice with 2 water and adjusted to pH 6.0. To this was added 0.5 mL of β-galactosidase (Lactress L3 manufactured by Daiwa Kasei Co., Ltd.) (16 U per gram of solid content), and the mixture was held at 53 ° C. for 2 hours. This was boiled to inactivate the enzyme, and activated carbon was inactivated by adding several grams of activated carbon. This was filtered with a 0.45 μm filter and then desalted with an ion exchange resin (Amberlite MB4 manufactured by Organo). This was concentrated under reduced pressure to give a solid content concentration of 30% (w / v) for resin fractionation (chromatographic separation).
For resin fractionation, an improved simulated moving bed chromatographic separation apparatus manufactured by Nippon Nersui Co., Ltd. was used. Fractionation conditions were as follows, and a differential refractometer (Atago PR-201) was used as a detector.
Sample injection volume 113mL (30% (w / v))
Column packing material Sodium-type strongly acidic cation exchange resin (UBK530 manufactured by Mitsubishi Chemical)
Column size φ28.0 x 550 mm (339 ml) x 4 Column temperature 80 ° C
Flow rate 11.3 mL / min Mobile phase Water Fractionation volume 15 mL (prepared immediately after sugar is detected in the eluate)

As a result, sugar was eluted in about 40 minutes after loading, and the monosaccharide fraction and the disaccharide fraction were well separated. The chromatogram of this chromatographic fraction is shown in FIG. As a result of collecting and concentrating fraction numbers 14 to 19 as the disaccharide fraction, high purity epilactose with a purity of 44.9% was obtained. The raw material solid content recovery rate was 40.9%.

[Example 2]
100 mL of 1M potassium phosphate buffer (pH 6.3) is added to 20 L of 10% (w / v) lactose monohydrate (manufactured by Kanto Chemical Co., Ltd.), and luminococcus albus-derived cellobiose 2-epimerase is added per 1 g of lactose solids. It added so that it might become 1U. This was held at 25 ° C. for 60 hours. When the epilactose content in the reaction solution after the enzyme reaction was measured by the sugar composition analysis method, the epilactose content in the total sugar solids was 26.2%. That is, the epilactose purity at this stage was 26.2%.

  Thereafter, several grams of activated carbon was added to the reaction solution after the enzyme reaction, followed by boiling treatment. Subsequently, it filtered with the 0.45 micrometer average pore diameter filter, and concentrated under reduced pressure until the solid content concentration became 70% (w / v). The obtained concentrated liquid was left overnight at room temperature to precipitate crystals. Next, the precipitated crystals were removed by filtration with a filter paper to obtain a honey solution. The filtered crystals were analyzed by HPLC and confirmed to be lactose. Moreover, the epilactose content in solid content became 67.6% by the said honey operation. That is, the epilactose purity at this stage was 67.6%.

The honey solution obtained above is diluted with water to 10% (w / v), 1/100 volume of 1M potassium phosphate buffer (pH 6) and β-galactosidase (Lactress L3 manufactured by Daiwa Kasei) Was added and maintained at 53 ° C. for 3 hours. This was boiled to deactivate the enzyme, subjected to activated carbon treatment and desalting treatment in the same manner as in Example 1, and then filtered through a 0.45 μm filter to a solid content concentration of 60% (w / v). Concentrated. Then, what diluted the said concentrate with the pure water so that it might become solid content concentration 20% (w / v) was used for the resin fraction (chromatography separation).
For resin fractionation, an improved simulated moving bed chromatographic separation apparatus manufactured by Nippon Nersui Co., Ltd. was used. Fractionation conditions were as follows, and a differential refractometer (Atago PR-201) was used as a detector.
Sample injection volume 113mL (20% (w / v))
Column packing material Sodium-type strongly acidic cation exchange resin (UBK530 manufactured by Mitsubishi Chemical)
Column size φ28.0 x 550 mm (339 ml) x 4 Column temperature 80 ° C
Flow rate 11.3 mL / min Mobile phase Water Fraction volume 11.3 mL (preparation immediately after sugar is detected in the eluate)

As a result, the monosaccharide fraction and the disaccharide fraction were well separated. The chromatogram of this chromatographic fraction is shown in FIG. As a result of collecting and concentrating fraction numbers 16 to 18 as a disaccharide fraction, a result of collecting lactose of no less than 90% with a 21% raw material solids recovery rate, and collecting and concentrating fraction numbers 12 to 19 An epilactose with a purity of 85% or less was obtained with a raw material solid content recovery of 53%.

[Comparative Example 1]
The enzyme reaction solution obtained by allowing cellobiose 2-epimerase to act on lactose in the same manner as in Example 1 was subjected to chromatographic separation in the same manner as in Example 1 without hydrolysis with β-galactosidase. Since lactose and epilactose were fractionated as sugar fractions, it was not possible to highly purify epilactose by chromatographic separation.

[Reference Example 1]
1 mL of 1M phosphate buffer (pH 8.4) and 50 U of cellobiose 2-epimerase were added to 100 mL of 10% lactose and kept at room temperature for 3 days. This was deactivated by boiling, and then desalted with an ion exchange resin (Amberlite MB4 manufactured by Organo) was filtered through a 0.45 μm filter. This was concentrated under reduced pressure and adjusted to each concentration, and kept at 4 ° C. for 3 days. This was filtered through a 0.45 μm filter to remove lactose crystals, and the epilactose content was measured by HPLC. (HPLC conditions were the same as in the cellobiose 2-epimerase activity measurement method). The results are shown in FIG. As shown in FIG. 3, the highest epilactose content was obtained when the reaction solution was concentrated to a solid content concentration of 63.6%. At this time, the epilactose content was 67.7%. At a concentration higher than this, the epilactose content was reduced, and when it was 68.7% or more, no lactose crystals were formed due to the inhibitory effect of crystallization by epilactose. From this, it was revealed that there is a limit to the achievable epilactose purity by simply crystallizing and removing lactose.

  The present invention is useful for producing epilactose applicable to food and drink.

Claims (7)

Allowing β-galactosidase to act on a solution containing lactose and epilactose;
Chromatographic separation of a monosaccharide fraction and a disaccharide fraction from the solution in which the β-galactosidase is allowed to act; and
Recovering epilactose from the disaccharide fraction,
The manufacturing method of the high purity epilactose characterized by including this. The method according to claim 1, further comprising subjecting the solution containing lactose and epilactose to a crystallization treatment and separating the precipitated crystallized product before allowing β-galactosidase to act. The production method according to claim 1 or 2, wherein the chromatographic separation is performed using an alkali metal type or alkaline earth metal type cation exchange resin. The said ion exchange resin is a manufacturing method of Claim 3 which is a sodium type cation exchange resin. The manufacturing method according to any one of claims 1 to 4, wherein the chromatographic separation is performed by simulated moving bed column chromatography. The manufacturing method of any one of Claims 1-5 which obtains the solution containing the said lactose and epilactose by the enzyme reaction of cellobiose 2-epimerase which uses lactose as a substrate. The high purity epilactose obtained by the method of any one of Claims 1-6.